Two-dimensional gel electrophoresis (2D gel electrophoresis) is a conventional method for separating, e.g., complex protein mixtures. This method typically consists of two electrophoresis steps that are carried out separately and successively, namely isoelectric focusing (IEF) (1st dimension) and denaturing SDS polyacrylamide gel electrophoresis (SDS-PAGE) (2nd dimension). IEF, as a rule, is performed in polyacrylamide gel strips or gel rods with immobilized pH gradients. Immobilized pH gradients are distinguished in that the buffer substances are covalently bound in the polymer; they no longer have to be applied together with the sample. The gradient is thus fixed within the gel. As an alternative thereto, normal polyacrylamide gel may be used if ampholytes are applied together with the sample. The fixed pH gradient is obtained by dipping the corresponding ends of the gel into an acid buffer (anolyte) and an alkaline buffer (catholyte). In a subsequently applied electric field, the ampholytes migrate into their specific isoelectric region and thereby establish and stabilize the pH gradient. The molecules to be analyzed migrate in the electric field to the position where the gradient has a pH value corresponding to the isoelectric point of the molecule. Thus the proteins are separated in this first direction irrespective of their size or mobility and exclusively based on their isoelectric properties. After completion of IEF and optional treatment in separate vessels with different mobilization and washing powders, the gel strips or gel rods are placed onto a typically vertical SDS-PAGE gel that is placed into an electrophoresis apparatus. When a voltage is applied, the proteins are transported out of the gel strip/gel rod of the 1st dimension and into the SDS-PAGE gel, where they are separated in a 2nd direction according to their molecular mass.
Both of these methods may in principle also be implemented in a capillary electrophoretic technique and are then referred to as CIEF and CGE, respectively.
After the first publication of a 2D electrophoresis method (P. H. O'Farrel 1975 J. Biol Chem. 250, 4007-4021), methods and devices were developed to allow the two methods to be carried out in a single apparatus or a single gel (J. Shevitz 1983: Electrophoretic system and method for multidimensional analysis; U.S. Pat. No. 4,385,974 and S. A. Hoefer 1978: Fluid isolation electrophoresis apparatus and method; U.S. Pat. No. 4,101,401). These documents already describe approaches for the parallel processing of a plurality of samples. With commercially available systems (Dalt system, Hoefer Scientific Instruments, San Francisco, Calif., USA) it is possible, for example, to subject up to 12 samples simultaneously to IEF and to run up to 10 SDS PAGE gels in a single operation. A clear miniaturization of these electrophoresis methods has thus far been achieved by making the gels smaller and by introducing a partially automated electrophoresis system.
During the last few years, the capillary electrophoretic methods have also been further developed using microsystem technology. The trend is to produce chips on which the electrophoresis elements are mounted in miniaturized form. Known in the art are the use of suitable chips for CIEF of proteins (O. Hofman, D. Che, K. A. Cruickshank & U. Müller 1999: Adaptation of capillary isoelectric focusing to microchannels on a glass chip. Anal. Chem. 71, 678-686 and J. Xu, L. Locascio, M. Gaitan & C. S. Lee 2000: Room temperature imprinting method for plastic microchannel fabrication. Anal. Chem. 72, 1930-1933) as well as the SDS gel based separation of proteins (S. Yao, D. S. Anex, W. B. Caldwell, D. W. Arnold, K. B. Smith & P. G. Schultz 1999: SDS capillary gel electrophoresis of proteins in microfabricated channels. Proc. Natl. Acad. Sci. USA 96, 5372-5377). Solutions are known, for example, which generally propose to combine two electrophoresis techniques that are orthogonal to one another, as described, for example, in U.S. Pat. No. 5,599,432.
It is generally known that conventional 2D gel electrophoresis is very time-consuming. The analysis of a sample can take up to approximately 2 days. Because of the many complex work steps involved, the method can be carried out only by specially trained technical personnel.
It has been found that automation is possible only to a limited extent. Partially automated commercially available systems (e.g. the Phast system) still require many manual work steps. The dimensions of the gel apparatuses that have been used until now permit parallel analyses only to a limited extent (approximately 10 samples) but no high throughput analyses.
U.S. Pat. No. 4,385,974 describes a two-dimensional separation method and an apparatus for carrying out successively both IEF and, perpendicularly thereto, SDS-PAGE without any gels or gel pieces having to be moved. An electrically non-conductive fluid barrier, preferably glycerin, is arranged between the IEF gel and the SDS-PA gel. After completion of the first IEF separation step, this fluid barrier is aspirated using a tip and is replaced with an equilibration buffer. This method, however, still requires many additional manual steps: casting and polymerizing the SDS-PA gel, covering with a layer of glycerin, casting and polymerizing the IEF gel, installing the gel cassette in the device, filling the reservoir, etc. As a result, this complex method precludes full automation.
The use of a single plate gel for both work steps as described in U.S. Pat. No. 4,101,401 leads to insufficient resolution of the protein bands because of the very different requirements to be met by the composition of the two separation matrices. Attempts have also been made to carry out multi-dimensional electrophoresis on a microscale basis. U.S. Pat. No. 6,013,165, for example, describes two devices. The first is suitable for the conventional combination of IEF and SDS-PAGE and is provided with a cavity, whereas the second contains a field traversed by a plurality of parallel microchannels and can be used for carrying out alternative 2D-methods. This document does not describe any approaches to automating the conventional method, which requires rebuffering or protein mobilization steps, among others.